The metabolism of proteins by microorganisms or other cellular entities is of particular interest in view of the known relationship of protein "turnover" rate and various physiological states. Since the synthesis and degradation of cellular and extracellular proteins are normally carefully regulated by the body, variations in the combined synthesis and degradation rates, termed "turnover" rate, may be diagnostic of normal or abnormal physiologic events. For example, changes in the turnover rate of heart mitochondrial or microsomal proteins may signify incipient disease, or induction or reduction of globins or enzymes which may be a response to unusual levels of regulating factors such as steroid hormones. Thus, methods for assessing synthesis and degradation rates of proteins are particularly desirable, both from the standpoint of identifying normal or abnormal inducing events and of relating turnover rate to specific disease states in diagnostic applications.
Radioactive labelling procedures are widely used as a means of measuring the metabolic activity of microorganisms. Specifically, the synthesis of various cellular constituents can be assessed by quantification of rates and amounts of incorporation of radioactive materials into the cellular constituents of microorganisms. Known radiochemical techniques are, however, insufficient by themselves to simultaneously quantify multiple protein turnover rates, as these techniques are generally inherently limited to a determination of the rater of either synthesis or degradation of a given molecule. For example, in an equilibrium labelling format, a steady state microorganism culture may be presented with a radioactivity labelled metabolite, and samples removed at stated intervals; the test molecule, such as protein, is then isolated and its radioactive content determined. While the resultant data reflects the rate of incorporation of the labelled metabolite into the synthesized test protein molecule, the procedure does not yield data sufficient to simultaneously determine turnover rate of specific proteins. Accordingly, when it is desired to evaluate protein turnover rate, radiolabelling is used in conjunction with procedures for resolving protein mixtures and determining the concentration of proteins of interest.